Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation

Abstract

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the β-casein gene promoter (p-344/-1βc-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction ( R = 0.99, P < 0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous β -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.